Showing papers in "Journal of Neurochemistry in 1963"

CHROMATOGRAPHlC SEPARATION OF HUMAN BRAIN GANGLIOSIDES

Abstract: GANGLIOSIDES are defined as acylsphingosyl oligosaccharides containing sialic acid (KLENK, 1942). For several years brain gangliosides were considered to contain a common carbohydrate moiety (KLENK and LAUENSTEIN, 1953 ; BOGOCH, 1958). The occurrence of brain gangliosides with different carbohydrate moieties was suggested (SVENNERHOLM, 1956, 1957a), since different ganglioside preparations varied in their composition and behaviour on cellulose columns. KUHN and EGGE (1959) confirmed the chromatographic separation of brain gangliosides and showed that the slowmoving gangliosides had a considerably more complicated structure than the fastmoving ones. During the last two years reports have appeared from many laboratories on the complexity of brain gangliosides. SVENNEMOLM and RAAL (1961) isolated monoand disialogangliosides from human brains, KUHN, WIEGANDT and EGGE (1961) found one inonosialoganglioside, two disialogangliosides and one trisialoganglioside in human and calf brains, and DAIN, et al. (1962) isolated four components from ox brain with similar composition to those of KUHN et al. (1961). KLENK and GIELEN (19614 isolated the hexosamine-containing and the hexosaniinefree monosialogangliosides originally postulated by SVENNERHOLM (19576). In a recent communication (SVENNERHOLM, 1962) it was shown that predominantly the gangliosides have a common basic structure : N-acyl-sphingosine-glucose-galactoseN-acetylgalactosamine-galactose to which one or more molecules of N-acetylneuraininic acid are bound. In the present report chromatographic methods for the quantitative isolation and separation of human brain gangliosides are described. These methods have been applied to studies of the ganglioside fraction of normal foetal, infant and adult brains while several different gangliosides have been isolated and further characterized. A knowledge of the normal ganglioside pattern is important in the study of the metabolism of the gangliosides but it is also necessary for the detection of disturbances in ganglioside metabolism. In infantile amaurotic idiocy (SVENNERHOLM, 1962) for example, there are no abnormal gangliosides but excessive amounts of a ganglioside are present which normally constitute only a few per cent of the total ganglioside fraction. It is likely that in several other mental or neurological disorders there is a disturbance of the normal ganglioside pattern. As this can now be determined by simple chromatographic methods it will be quite easy to detect minor deviations from the normal ganglioside metabolism.

Isolation of synaptic vesicles and structural organization of the acetylcholine system within brain nerve endings

Abstract: THE DISCOVERY of synaptic vesicles as the most significative component of nerve endings (DE ROBERTIS and BENNETT, 1954, 1955) led to the suggestion that they could be the site of storage and synthesis of transmitter substances. Since then this concept has become associated with the notion of a “quantized” release of acetylcholine at the neuromuscular junction (DEL CASTILLO and KATZ, 1956). At the same time several indirect indications of the role of these vesicles in synaptic transmission were demonstrated (DE ROBERTIS, 1959). The stimulation of some cholinergic terminals by DE ROBERTIS and VAZ FERREIRA (1957) and subsequent electronmicroscope observations suggested that within the endings a balance exists between the formation and release of synaptic vesicles, related to the frequency of stimulation. The isolation of such a subcellular component remained as the most direct approach to the elucidation of the chemical nature and physiological significance of synaptic vesicles. An approach to such a goal was made independently by DE ROBERTIS et al. (1960, 1961a) and GRAY and WHITTAKER (1960) with the demonstration that the so-called mitochondrial fraction of the brain contained a considerable number of isolated nerve endings in addition to free mitochondria and myelin. In our first paper we reported an attempt to isolate synaptic vesicles by disrupting the endings mechanically and subfractionating the microsomes in a continuous gradient. However the vesicles isolated in one of the four microsomal fractions were not as regular in size as the true synaptic ones. At that time we postulated that: “the further purification of intact synaptic terminals may provide a good starting point for the isolation of pure synaptic vesicles”. We also stated that : “when this is done, the synaptic vesicles will be probably found in one of the submicrosomal fractions”. More recently a technique was developed in our laboratory which allows the subfractionation of the mitochondrial fraction (Mit)? into five separated layers which in order of increased density are essentially composed of: A, myelin; B, fragments of endings and membranes; C , cholinergic nerve endings; D, non-cholinergic nerve endings; and E, free mitochondria (see methods and electronmicrographs in DE ROBERTIS et al., 1962a). The two subfractions C and D of nerve endings provided an excellent material for the isolation of synaptic vesicles. However it was observed

Alteration of the fatty acid composition of brain lipids by varying levels of dietary essential fatty acids.

Abstract: THE FATTY acid composition of brain lipids is known to be relatively constant, not responding as readily to changes in dietary fat composition as do the lipids of other tissues. This has been considered to be due to the blood-brain barrier which is thought to restrict the passage of fatty acids into the brain. This concept is based upon several investigations of the metabolic behaviour of fatty acids of brain lipids. WAELSCH et al. (1940, 1941) found that deuterium labelled fatty acids were incorporated into brain lipids only in traces, whereas incorporation into other organ lipids was significant. When KLENK (1957) fed [14C]acetate to rats, saturated as well as monoenoic and polyenoic fatty acids of brain-lipids showed a significant uptake of radioactivity. However, the levels were much lower than those found in liver lipids. Incubation of brain slices with [I4C]acetate led to 14C-incorporation comparable to that in liver lipids. [14C] acetate injected intraperitoneally into rats was incorporated into cholesterol and fatty acids of brain in almost negligible, but still detectable, amounts (NICHOLAS and THOMAS (1959)). A high yield of I4C in lipids was observed when the [lelacetate was injected intracerebrally, thus bypassing the blood-brain barrier. However, WITTING and co-workers (1961) have noted changes in the concentrations of linoleic, arachidonic and docosahexaenoic acids of brain mitochondria by feeding different amounts of corn-, coconut-, and codliver oils to rats. In a series of recent investigations MOHRHAUER and HOLMAN have described the pronounced changes in the fatty acid composition of liver (1963~) and heart lipids (1963b) effected by changing dose levels of essential fatty acids (EFA)$. Furthermore, they have studied the metabolic interrelationship of dietary linoleic and liholenic acids (1963~) in feeding experiments. Those experiments on the effect of dietary EFA upon heart and liver lipids offered an opportunity to make a similar study of the variations in brain fatty acids. E X P E R l M E N T A L Substances: The ethyl linoleate and ethyl linolenate were obtained from The Horniel Foundation, Austin, Minnesota. GLC analysis indicated the linoleate to be 98.3 per cent pure. The only contaminant was 1.7 per cent oleate. The linolenate was found to be 97.3 per cent pure. It contained less than 0.4 per cent linoleate and five other minor unidentified impurities on GLC analysis. The ethyl

A controlled study of enzymic activities in three human disorders of glycolipid metabolism.

Abstract: A CONTROLLED STUDY OF ENZYMIC ACTIVlTIES IN THREE HUMAN DISORDERS OF GLYCOLIPID METABOLISM” JAMES H. AUSTIN Division of Neurology, University of Oregon Medical School, Portland, Oregon, U.S.A. A . s. BALASUBRAMANIAN~, T. N. PATTABIRAMAN$, s. SARASWATHI, D. K. BASUS and B. K. BACHHAWAT Neurochemistry Laboratory, Department of Neurology and Neurosurgery, Christian Medical College & Hospital, Vellore, S. India

Free amino acids and related compounds in dog brain: post-mortem and anoxic changes, effects of ammonium chloride infusion, and levels during seizures induced by picrotoxin and by pentylenetetrazol.

Abstract: PREVIOUS studies in this laboratory have been concerned with chemical changes in the brain associated with convulsive activity (STONE et al., 1960) as revealed by analysis of brain tissue after in vivo fixation with liquid air. The development by MOORE and STEIN (1954) of ion exchange procedures for the separation of amino acids has made it feasible to extend the study of cerebral constituents during convulsions to include many free amino acids and related substances. These compounds are of particular interest in view of recent investigations by GEIGER and his coworkers (1960 a, 6) indicating that protein turnover in the brain is accelerated during convulsive activity induced by pentylenetetrazol. Accordingly, the cerebral levels of a large number of nitrogenous constituents have been determined in control experiments and during seizures induced by picrotoxin or by pentylenetetrazol. In an attempt to classify different types of seizures on the basis of specific chemical patterns (STONE et al., 1960), the seizures induced by these two agents have been tentatively classed together as representing a type in which distinctive metabolic changes have not yet been observed. As an aid in the interpretation of any changes which might be observed during seizures or other conditions, it was considered essential to study also post-mortem changes and the effects of anoxia and of ammonium chloride infusion.

The metabolism of [c14]noradrenaline by cat brain in vivo*

Abstract: A WIDE variety of effects on the central nervous system has been attributed to catecholamines, yet information on the metabolic fate of noradrenaline in brain is still based largely on indirect evidence. Previous studies on the metabolism of adrenaline and noradrenaline have shown that little, if any, of these compounds traverses the blood brain barrier from the systemic circulation (DE SCHAEPDRYVER and KIRSHNER, 1961; WEIL-MALHERBE et al., 1959). The enzymes chiefly responsible for the immediate inactivation of catecholamines in other tissues, catechol-0-methyl transferase (COMT): (AXELROD et al., 1959) and monoamine oxidase (MAO) (BHAGVAT et al., 1939; BLASCHKO et al., 1937; BOGDANSKI et al., 1957) have been shown to be present in the brain, but their relative importance in the inactivation of catecholamines in this tissue has not been fully evaluated. CROUT et al. (1961) and WEIL-MALHERBE et al. (1961) have presented evidence indicating that M A 0 is chiefly responsible for the inactivation of endogenously released catecholamines in rat heart and rat and rabbit brain. GOLDSTEIN et al. (1959), using homogenates of cow brain have shown that M A 0 activity could account for most of the metabolites of 3-hydroxytyramine. The studies reported here, however, indicate that COMT plays the predominant role in the inactivation of adrenaline and noradrenaline injected into the lateral ventricle of the cat.

Active transport of amino acid into cerebral cortex slices.

Abstract: IN GENERAL, in nerve or muscle the functions of cations like sodium and potassium are very closely related to permeability changes of cell membranes (HODGKIN, 1951). As is well known, some amino acids such as glutamic acid, aspartic acid and y-aminobutyric acid (GABA)~, which are thought to have special functions in the central nervous system in mammals, exist in considerably higher concentrations in the brain than elsewhere. When brain cortex slices were incubated aerobically at 37\" in saline containing these amino acids, they were actively accumulated in the brain cells in the presence of glucose. As reported before (TERNER, EGGLESTON and KREBS, 1950), L-glutamate added to the medium is accumulated actively in brain slices during incubation. This paper presents a biochemical background for the active transport mechanism by which these amino acids are accumulated across the brain cell membrane.

Effects of temperature, calcium and activity on phospholtpid metabolism in a sympathetic ganglion*

Abstract: THE MAJOR purpose of these experiments was to investigate the changes in phospholipid metabolism which occur when nerve cells are activated by naturally conducted impulses. For this purpose, impulses were initiated under experimental control at a point effectively remote from that at which the metabolic effects of conduction and synaptic transmission were measured (Fig. 1). Observations such as those of

THE α‐AMINOBUTYRIC ACID AND FACTOR I CONTENT OF BRAIN *

Abstract: IN AN earlier report from this laboratory (LEVIN, LOVELL and ELLIOTT, 19616) it was contended that the y-aminobutyric acid (GABA)? content of brain, as determined by a paper chromatographic-ninhydrin method, could account for all the Factor I activity of brain extracts. We define Factor I as a natural substance which inhibits the spontaneous activity of the crayfish stretch receptor neuron. Since reports have appeared in which it is claimed that brain (MCLENNAN, 1959) and crayfish inhibitory nerve (FLOREY and BIEDERMAN, 1960; FLOREY and CHAPMAN, 1961 ; see, however, KRAVITZ, POTTER and VAN GELDER, 1962) contain Factor I activity that cannot be accounted for by GABA, it seemed advisable to establish the contention of LEVIN et al. (1961 6) more rigorously. In the present report Factor I activityin various parts of beef brain has been compared with the GABA content. It has been observed previously that the Factor I content of brain tissue increases with time after excision (ELLIOTT and FLOREY, 1956). In the present study it is shown that the GABA content of excised brain can increase by 100 per cent or more during standing at room temperature. This increase can be prevented by keeping the tissue cold. If the brain is frozen in situ in liquid air at the moment of death, and excised and extracted while still frozen, considerably less GABA is found than if the tissue is excised and extracted rapidly without freezing. In view of this evidence of a rapid post-mortem increase in the GABA content of the brain, the effects of agents which have previously been reported to affect GABA levels have been re-examined. Preliminary reports of some of these findings have been published elsewhere (LEVIN et al., 1961a; ELLIOTT and LOVELL, 1962).

Enzyme inactivity of myelin: histochemical and biochemical evidence.

Abstract: EARLIER work on the lipid and protein metabolism of grey and white matter of brain suggested that the myelin sheath may be in a metabolically dynamic state (SCHEINBERG and KOREY, 1962). Quantitative cytochemical studies of Ammon’s horn in the rabbit (LOWRY et af., 1954) first suggested that myelin may itself be enzyinically active. This view was based on finding more enzyme activity and more acid soluble phosphorus derivatives in the myelinated layer than would then be expected from the content of axons and neuroglia. More recently, it has been claimed that a wide range of oxidative and hydrolytic enzymes are present i n the myelin sheath of the peripheral nerve and in its neurokeratiri network (WOLFGRAM and ROSE, 1960; TEWARI and BOURNE, 1960a, b and c): some enzyme activity has also been reported in CNS myelin (TEWAIU and BOURNE, 1962n and h). TEWARI and BOURNE regard the tieurokeratin network as the locus of metabolic activity within the myelin sheath. If these histological and histochemical observations on neurokeratin and myelin enzymes can be substantiated, the myelin sheath would appear to be potentially metabolically active and could be regarded as a functional part of the cytoplasm of its formative cell-oligodendrocyte or Schwann cell~-and therefore, susceptible to injury by metabolic derangements. However, evidence has been accumulated from isotope studies to show that myelin lipids (DAVISON et al., 1959 a and 6; DAVISON and D o B B I N c ; , 1960) and proteins

Analysis of the somato-axonal movement of phospholipids in the vagus and hypoglossal nerves.

Abstract: WEISS and HISCOE (1948) reported that the axoplasm of the peripheral nerve fibre is maintained in constant proximo-distal motion. By analysing the effects of the temporary reduction of the fibre diameter on the column of axoplasm, they obtained evidence that axoplasm moves towards the periphery at a rate of approximately 1 mm per day. WEISS and HISCOE (1948) in addition, postulated that the source of the axonal constituents is in the nucleated cell space and that the proximo-distal convection of the neuroplasm “serves to replace catabolized protoplasmatic systems which cannot be synthesized in the peripheral cytoplasm”. The primary object of most of the observations hitherto reported is to obtain direct evidence of the hypothesis of WEISS and HISCOE (1948). Three methods of investigation have been especially employed : (1) Radioactive tracer methods ; (2) Analyses of neuroplasmic enzymes; (3) Observations on isolated nerve cells in tissue culture. SAMUELS et al. (1951), WAELSCH (1958), KOENIC (1958) and LAJTHA (1961) have tried to trace the movement of the axoplasm by measuring the concentration of isotopically labelled phosphoprotein and protein at different levels in the sciatic nerve after administration of r2P]phosphate, [14C]lysine and rjslmethionine, respectively. OCHS et al. (1962) have taken a succession of measurements of radioactivity along the length of the lumbar ventral roots after injection of 32P into the lumbar spinal cord of cats. Investigations by these methods appeared to show an initial rather steep proximo-distal gradient of radioactivity, which flattened and eventually equalized as time went on. Notwithstanding this progress, however, it has remained undecided (i) whether tagged material in downward transit along nerves was inside or outside the axons and (ii) whether the source of the labelled proteins and phosphoproteins was central, i.e. in the perikaryon or peripheral, i.e. in the Schwann celIs and connective tissue cells. Studies for testing the hypothesis of WEISS and HISCOE (1948) have been also centred on the acetylcholinesterase (ATChE) (SAWYER, 1946) and choline acetylase (ChAc)* (HEBB and WAITES, 1956; HEBB and SILVER, 1961). In both the studies by these authors the increase of AChE and ChAc in front of a cut nerve, and the concomitant steep fall of those in degenerating stumps have been regarded as direct evidence for enzymic movements from the central area into the periphery. In contrast to the above, KOENIG and KOELLE (1961) as well as CLOUET and WAELSCH (1961) observed that the restoration of AChE activity proceeded in a distal-proximal direction after the irreversible inactivation of the enzyme by organophosphorus

A comparative mapping of enzymes involved in hexosemonophosphate shunt and citric acid cycle in the brain.

Abstract: REPORTS of preceding investigations (FRIEDE, 1959~2, 1961 c; FRIEDE and FLEMING, 1962) have contained detailed charts of the distribution of several enzymes in the brain. The purpose of this paper is to compare enzyme patterns with each other and to describe similarities and, particularly, differences of the patterns that were observed. The regional patterns in the brain of seven enzymes which are concerned with glucose metabolism were studied and compared. Most of the data were obtained from histochemical series from rhesus monkey and human brains. These were compared with tissue homogenate assays. The enzymes studied were cytochrome oxidasef succinic dehydrogenase, DPN-diaphorase, TPN-diaphorase, malic dehydrogenase glucose-6-phosphate dehydrogenase, 6-phosphogluconic dehydrogenase and lactic dehydrogenase. In order to render this article more complete, some previously published data have been included. MATERIALS AND METHODS Because of difficulty in obtaining adequate human material, the systematic work reported in this study was done on rhesus monkey brains. Most of the Observations were compared with observations on human material as it became available during the course of the study. The following material was used: Monkey: Microscopic histochemicai surveys w2ere made by series for LDH, G-6-P, GPD, DPN-diaphorase, TPN-diaphorase, CYO and SD. Extensive spectrophotometric measurements of LDH were made from histochemical sections. SD, DPN-diaphorase, and G-6-P were assayed in homogenates from various regions and the data were compared with the histochemical staining gradients. Plans to assay all the nuclei of the monkey brain had to be discarded because the small size of the brain did not permit accurate macroscopical sampling of smaller nuclei. Random histochemical material was available for SD, LDH, CYO and G-6-P. The distribution of DPN-diaphorase was available from previous mappings (FRIEDE and FLEMING, 1962). In addition, G-6-P, DPN-diaphorase and SD were assayed in homogenates of seventeen representative regions from five normal human brains obtained 3.5 to 10 hours after death. The assay measurements were used for comparison with the histochemical gradations, as well as for comparison with each other (Table 1, line 6). Man: Also included are some observations on the distribution of MDH in the cat brain stem. Methods : Complete details of methodology are given in a separate publication (FRIEDE, FLEMING and KNOLLER, 1963) in which histochemical methods were tested extensively by comparison with assays in tissue homogenates. At this time, it may suffice to indicate that the use of formalin-fixed material was an advantage for histochemical studies of DPN-diaphorase and LDH. When carried out under carefully controlled conditions, fixation prevented loss of these two enzymes from tissue blocks and sections; histochemical data obtained by spectrophotometric measurement of formazan were in excellent agreement with the data of assays of unfixed tissue homogenates. Attempts to elaborate and standardize fixation methods for the other enzymes studied have failed so far.

Studies on brain lipids in Tay-Sachs' disease. I. Isolation of two sialic acid-free glycolipids.

Abstract: A NUMBER of glycolipids (in addition to cerebrosides) containing sphingosine, fatty acids and sugars (glucose, galactose and/or hexosamine) have been isolated from spleen and erythrocyte stroma of various species (KLENK and DEBUCH, 1959). Although lipids of this composition have not been identified with certainty in brain tissue, there is nevertheless some indication that they might be present in small amounts (WEISS, 1956; KLENK et al., 1957; SVENNERHOLM and RAAL, 1961). Two such glycolipids have now been isolated from brain tissue of patients with Infantile Amaurotic Familial Idiocy (Tay-Sach's disease). The main biochemical abnormality which characterizes this disease is an accumulation of gangliosides (KLENK, 1939); the two new compounds which have now been identified in brain tissue of Tay-Sachs' disease belong to the same class of lipids. The isolation and chemical composition of these two glycolipids are described in the present communication. Some of this work has appeared in preliminary reports (GATT and BERMAN, 1961 ; BERMAN and GATT, 1962).

The action of neuraminidase from clostridium perfringens on gangliosides.

Abstract: GANGLIOSIDES from brain tissue are chloroform-methanol soluble, water soluble, nondialysable glycolipids which contain N-acetyl neuraminic acid. Part of the NANAT is labile to dilute acid and enzymatic hydrolysis (FOLCH-PI and LEES, 1959; KLENK and GIELEN, 1960; KUHN et al., 1960). Clostridiumperfringens culture filtrate has been previously reported to hydrolyse the NANA from glycoproteins such as orosomucoid from blood serum (POPENOE and DREW, 1957). This paper reports the characteristics of this C. perfringens exoenzyme and its action in cleaving part of the NANA from ganglioside preparations. lnvestigations with this enzyme as a tool in the study of ganglioside structure and biosynthesis will be described in subsequent work.

The brain barrier system. v. stereospecificity of amino acid uptake, exchange and efflux.

Abstract: A NUMBER of studies including some from our laboratory provide evidence that the uptake of amino acids by the brain in civo occurs mainly through mediated transport (for a recent review see LAJTHA, 1962). Recently evidence for similar transport was found also in the exit of amino acids from the brain (LAJTHA and TOTH, 1961). Since the cerebral levels of a compound will be determined by mechanisms of efflux as well as influx, it is of interest to establish the relationship of these fluxes in the two directions. It is of obvious theoretical and practical importance to establish the role that mechanisms of exit play in cerebral homeostasis. These studies were initiated as an attempt to find out whether or not the same transport mechanism participates in the uptake as that which participates in the efflux of amino acids in the brain. Because few direct approaches seem to be available for the living animal, a comparison of various properties of the two fluxes were chosen, the present paper being a comparison of the stereospecificity of the fluxes. It is hoped that comparison of the non-metabolizable with the natural analogue will elucidate the possible role of the metabolism of a compound in its uptake. The degree of stereospecificity in the transport of the various amino acids gives important information about the nature of the carrier, or carriers. If it can be shown in addition that the stereoisomers of a compound are specific for the same carrier or for the same transport mechanism, then the comparison of the stereospecificity of uptake with that of efflux may answer the question of whether uptake is mediated by the same or by a different mechanism than efflux.

Diamine oxidase in the brain of vertebrates.

Abstract: THE BRAIN of mammals (e.g. rat, ox, hog) contains relatively high amounts of diamines, e.g. spermine, spermidine and putrescine (DUDLEY et al., 1924; ROSENTHAL and TABOR, 1956; KEWITZ, 1959). Little is known about metabolism and function of these amines. Low activity of a diamine-metabolizing enzyme, diamine oxidase (diamine: 0, oxidoreductase, 1.4.3.6.) (DAO)?, has been found in brain of humans and mammals (monkey, rabbit, guinea pig, rat, mouse). These findings might, however, not be conclusive, since DAO was determined either by manometry, an insensitive method (ZELLER et al., 1939; BIRKHAUSER, 1940); or by the disappearance of histamine (COTZIAS and DOLE, 1952; MATSUI et al., 1959; SATO, 1959; VERSTER et al., 1959), which is not a specific substrate of this enzyme (SCHAYER, 1959). Therefore, in the present study the occurrciice of DAO in the brain of various vertebrates has been reinvestigated with a newly described, relatively specific and sensitive method using 14C-labelled putrescine and cadaverine as substrates. The activity of the enzyme was compared with that of monoamine oxidase (MAO) and of the aromatic amino acid decarboxylase (DCO). METHODS The substrates [l ,5-14C]cadaverine dihydrochloridet or [I ,4-14C]putrescine dihydrochloride (New England Nuclear Corp.) were diluted with the corresponding unlabelled diamines to a final concentration of 1 5 8 p ~ / m l and an activity of 0~05pc/ml. The brains of various animal species were immediately removed after decapitation, blotted on filter paper, and homogenized at +2". DAO activity was measured according to the slightly modified method of KOBAYASHI and OKUYAMA (1961). Homogenization was performed with an Oinnimixer (16,000 rev/min) in ~ / 1 5 Na/K-phosphate buffer pH = 7.25 (10-100 ml/g fresh weight). After saturation of the homogenate with noctanol (in order to inhibit M A 0 (HEIM, 1950)) and centrifugation at +2" and 29,000 g for 30 min, 0.5-2 ml of the supernatant were incubated with 1 ml of substrate solution (containing substrate in excess) under air at 37.5" for 1-6 hrs. Thereafter, the incubation mixture was shaken with 0.3 g of NaHCO, at room temperature for 5 min and subsequently extracted with 15 ml of toluene counting solution (4%, 2,5-diphenyloxazole and 15%" 1,4 bis [2-(diphenyloxazolyl)]-benzene). After phaseseparation (at +2" for about 30 min), 10 ml of the extract were counted in a Packard Tricarb liquid scintillation spectrometer. Non-incubated samples served as blanks. With this procedure, amounts as small as 0.001 pmole of diamine per g fresh tissue per hr can be detected with satisfactory accuracy. This is approximately 1000 times less than with Warburg manometry. ['4C]cadaverine gave generally the same results as [14C]putrescine. M A 0 activity was estimated in total homogenates (2-10%) in ice-cold ~ / 1 5 phosphate, pH = 7-25 (Teflon Potter-Elvehjem homogenizer) by manometry under Oa with 0.009 M-tyramine (final concentration) as substrate as described earlier (GEY and PLETSCHER, 1961). By this method, activities as low as 1.0 pmole O,/g fresh tissue/hr can be measured. D C 0 activitywas determined in 2 ml of supernatant as for DAO, hut without octanol and at pH 8.0 ( ~ / 1 5 Na/K-phosphate buffer). Incubation was performed under Na at 37.5' for 1 hr after addition of 0.5 pmole-5-hydroxytryptophan as substrate and 0.038 pmole-pyridoxal-5'-phosphate, followed by

Membrane potentials in isolated and electrically stimulated mammalian cerebral cortex. effects of chlorpromazine, cocaine, phenobarbitone and protamine on the tissue's electrical and chemical responses to stimulation

Abstract: THE TEST systems available for characterizing and understanding the actions of added substances on the mammalian central nervous system have been extended in recent years by using isolated cerebral tissues in two ways. (i) Metabolic sequelae to electrical stimulation of the tissue have been measured and found susceptible to agents not affecting the resting metabolism of the tissue (MCILWAIN, 19616; MCILWAIN and RODNIGHT, 1962). (ii) Resting membrane potentials have been measured in tissues maintained in isolation, and found sensitive to change in composition of bathing fluids (LI and MCILWAIN, 1957; HILLMAN and MCILWAIN, 1961). A further development is now reported in which displacement and recovery of membrane potential as a result of electrical stimulation are measured; the displacement and recovery have been found susceptible in varying degrees to chlorpromazine, phenobarbitone and protamine. To understand the effects of these substances, the N a and K have been determined in tissues electrically stimulated in the presence of several of the agents. Also during these studies on penetrating the tissue with microelectrodes, spike discharges similar to those observed by LI and MCILWAIN (1957) have been photographed and made the subject of systematic examination.

The effect of nervous activity on ribonucleic acid of the crustacean receptor neuron.

Abstract: APPROPRIATE stimulation of an experimental animal can cause histological changes in its nerve cells; cytoplasm, nuclei, and nucleoli may swell, shrink, or alter their staining properties (for review, see HYDEN, 1960). Changes in the quantity of protein and RNA* have been demonstrated by ultraviolet microspectrography (HYDEN, 1943; HMERGER and HYDBN, 1949a and b; ATTARDI, 1957). These reactions have been interpreted to reflect an increased turnover of protein and RNA. The variability of the results could be explained, if it were supposed that stimulation induces an increase of both synthesis and breakdown of protein and RNA in the nerve cell and that the particular physiological conditions determine which process predominates. The quantitative interpretation of ultraviolet absorption measurements on sectioned tissue is difficult (BRATTG~RD and HYDBN, 1952) but more recently RNA changes have also been demonstrated by quantitative microchemical methods (HYDBN and PIGON, 1960; HYDBN, 1963). At present it is impossible to decide if changes of RNA in the nerve cell are induced by increased nervous activity, as judged by a greater number of action potentials generated and conducted, or by modifications of the general metabolism of the animal due to the experimental conditions. In fact, considerable cytochemical reactions in nerve cells have been recorded shortly after administration of certain substances without noticeable signs of altered nervous activity (EGYHAzI~~~HYDBN, 1961 ; HYDEN and EGYHAZI, 1962). Furthermore, motoneurons stimulated antidromically have shown no changes in their ultraviolet absorption (LIu, BAILEY and WINDLE, 1950). The object of the present investigation was to examine the effect of nervous function, as defined by maintenance of membrane potential and generation and propagation of action potentials, on amount and base composition of neuronal RNA. The nerve cell of the isolated crustacean stretch receptor organ seemed to offer an ideal material for this purpose.

Isolation from rat brain of mitochondria devoid of glycolytic activity.

Abstract: BRAIN tissue is composed of a variety of cell types, ranging in diameter size from a few, to over 100, micra. Even inore significant is the fact that the protoplasmic bulk of brain is largely due to dendritic, axonal, and glial processes which intertwine about the diverse cellular elements. Although homogenization will readily rupture the cell bodies of larger neurons and glia, numerous processes and smaller cellular elements may be only partly disrupted, even after extensive homogenization or even ultrasonic disintegration. When conventional techniques of differential centrifugation are used in the isolation of mitochondria and other cellular elements from brain, it is extremely difficult to obtain reasonably homogeneous fractions, since many of the partly disrupted processes and cellular elements have similar sedimentation constants. Although brain mitochondrial preparations obtained by conventional methods are adequate for many purposes, they may often yield results which are misleading. The many reports that such preparations exhibited glycolytic activity (HESSELBACH and D u s w , 1953), have led some observers (BALAZS and RICHTER, 1958 ; GALLAGHER et al., 1956), to conclude that brain mitochondria contain glycolytic enzymes not present in liver or heart mitochondria. In one such study ABOOD et al. (1959) indicated that with hypertonic sucrose it was possible to obtain a fraction rich in mitochondria but almost devoid of glycolytic activity. The present study is devoted to a closer re-examination of the problems of the source of glycolysis in the mitochondrial fractions of rat brain. A more refined procedure for preparing relatively pure mitochondria as well as other cellular entities from rat brain is described. M E T H O D S

Postnatal changes in amino acid content of kitten cerebral cortex.

Abstract: THE VALUE of ontogenetic studies of the nervous system derives primarily from the possibility of correlating physiological development with structural and biochemical changes. The work of the FLEXNERS (cf. FLEXNER, 1955) on the developing guinea pig brain may be cited as an example of studies designed to provide such correlations. The present report initiates a series of biochemical investigations on the developing cat brain. The major objective sought in these studies is to specify metabolic factors which are associated with morphological (NOBACK and PURPURA, 1961 ; VOELLER, PAPPAS and PURPURA, 1963) and physiological (PURPURA, CARMICHAEL and HOUSEPIAN, 1960; PURPURA, 1961; PURPURA, 1962) maturation of the feline cerebral cortex. This study is concerned with postnatal changes in the brain content of glutamic and aspartic acids, glutamine, y-aminobutyric acid (GABA) f and glutathione (GSH). The results indicate that these compounds attain mature levels at different times. These temporal differences are discussed in relation to the possible distribution of amino acids in different parts of the neuron.

Distribution of acetylcholine in the brain during various states of activity.

Abstract: EARLIER work by HEBB and WHITTAKER (1958) and WHITTAKER (1959) indicated that the portion of brain acetylcholine (ACh) which is associated with the crude mitochondrial fraction, accounts for up to 70 per cent of all bound ACh which can be recovered from the whole homogenate. However some of the ACh present in the mitochondria1 preparations was not so stable as the remainder. Only about one-half of it could be released from the bound state by certain treatments, such as hypotonic dilution, while the rest was not affected. It is possible that this difference in binding reflects some difference in the state of the ACh in living brain tissue. In order to obtain more information about this, we have studied the subcellular distribution of ACh in mice and the effects of convulsions and anaesthesia on the bound ACh of the brain. A special strain of convulsive mouse (ep mouse), first described by IMAIZUMI et al. ( I 959), in which the brain ACh metabolism is apparently abnormal (KATO, 1959; KUROKAWA, KATO and MACIIIYAMA, 1961) was mainly used in this study.

The n-alkyl group specificity of choline acetylase from rat brain*†

Abstract: BECAUSE of the importance of acetylcholine in nerve transmission, much attention has been given to the occurrence and distribution of choline acetylase, the enzyme responsible for the synthesis of acetylcholine in nervous tissue. The enzyme, however, has not been extensively studied and relatively little is known regarding its substrate specificity (See the review by HEBB, 1957). Limited studies regarding the acyl group specificity of the enzyme obtained from various vertebrate and invertebrate sources have been reported (KOREY et ul; 1951 ; BERMAN et ul; 1953; GARDINER and WHITTAKER, 1954; REISBERG, 1957; FRONTALI, 1958; BERRY and WHITTAKER, 1959). The present work is concerned with the N-alkyl group specificity of the enzyme from rat brain, as studied with various analogues of choline.

Gamma-aminobutyric acid and oxygen poisoning*

Abstract: INTEREST in the problems associated with the breathing of OHPt has greatly increased with man’s progressive penetration to greater depths below the surface of the sea. The depth at which pure oxygen can be employed in diving operations is limited by the toxicity of the gas at high pressures (BERT, 1878). Symptoms of the toxicity are respiratory at oxygen pressures of 0.8 to 2.0 atmospheres but central nervous system symptoms predominate at higher pressures (STADIE et al., 1944). Although many years have elapsed since BERT’S initial studies on the subject the cause of the toxicity has eluded exact delineation. Possible explanations include interference with carbon dioxide transport (GESELL, 1923), changes in cerebral circulation (BEAN, 1945) and direct oxidation of cerebral enzymes (BEAN and HALDI, 1932). Mammalian brain contains considerable amounts of GABA (AWAPARA et al., 1950; ROBERTS and FRANKEL, 1950; UDENFRIEND, 1950), and it has been suggested that this amino acid may play an important role in some aspects of the regulation of physiological activity in the brain (KILLAM and BAIN, 1957). The concentration of GABA in the brains of rats during maximal seizure following hydrazide administration is only 70 per cent of the level observed in normal animals (KILLAM e l al., 1960), and ROBERTS and FRANKEL (1951) demonstrated that hydrazides inhibit the production of GABA by brain in vitro by interfering with glutamic acid decarboxylase. DASGUPTA et al. (1958) were able to prevent the onset of hydrazide-induced convulsions in cats by administering GABA. In view of these reports, the possibility that hydrazides induce convulsions in animals by causing a derangement of brain GABA metabolism cannot be dismissed completely. Since preliminary work in this laboratory (Wood and WATSON, 1962) indicated that administration of GABA to rats afforded the animals some protection against the toxic effects of OHP, the possibility must be considered that here too a deranged GABA metabolism is responsible for the observed disturbances in the functioning of the central nervous system. An investigation of this aspect of the problem is currently being carried out in our laboratories and the initial part of this study, concerning the protective action of GABA against oxygen toxicity and the possible correlation between the degree of protection and GABA levels in the tissues, is described in this paper.

The entry of cholesterol into rat brain during development.

Abstract: PREVIOUS experiments have shown a remarkable persistence of cholesterol laid down at the time of myelination in the chick and rabbit brain. [4-14C]Cholesterol was injected into the yolk sac of day-old chicks and intraperitoneally into 17-day-old rabbits (DAVISON, et al., 1958, 1959). It was recovered from the brain up to one year later, labelled in the same position and at no other (DAVISON and WAJDA, 1959a). An incidental finding in these experiments was that the cholesterol molecule itself could enter the developing brain, although brain cholesterol had hitherto been considered to be entirely derived from synthesis within the organ. This new finding has recently been challenged in experiments with rats (MORRIS and CHAIKOFF, 1961). In the present experiments rats have again been used in case there should be an unexpected species difference. The experiments were undertaken to determine (a) whether cholesterol as such could enter the brain; and if so (b) whether its rate of entry was as dependent on the timing of myelination as is the entry of other myelinsheath constituents and their precursors. A preliminary communication of a part of this work has already appeared (DOBBING and SANDS, 1963).

The calcium content of isolated cerebral tissues and their steady-state exchange of calcium.

Abstract: THE MONOVALENT ions of mammalian nervous tissues have been investigated extensively but the divalent ions of cerebral tissues have not received much attention. In neural tissues, the majority of the work on the divalent ions, and in particular calcium, has been carried out with frog and invertebrate nerves. The generalisations derived from these investigations may be suinmarised as follows: (a) sodium and potassium are the principal carriers of current across the membrane and their exchange accounts for the action potential; (b) calcium effects, primarily, the constraints upon these ionic movements (BRINK, 1954). The constraints upon the ionic movement probably are brought about by alterations in the membrane because variation in the tissue calcium causes changes in the electrical resistance of the membrane (HODGKIN and HUXLEY, 1952) and the calcuim ion concentration in the bathing solution affects the permeability of the cellular membrane (FRANKENHAEUSER and HODGKIN, 1955). Similarly, the threshold, the amplitude of action potential and rectification in frog nerves vary with the calcium concentration of fluids in which they are bathed (FRANKENHAEUSER, 1957). Metabolic changes have been reported with frog nerve preparations (GERARD, 1932) and with mammalian brain slices (GORE and MCILWAIN, 1952) in calcium deficient media. The processes underlying such metabolic changes are as yet only partly understood, but the changes have been attributed to participation of calcium in mechanisms linking the electro-chemical processes of conduction to the rate of oxidative metabolism (GERARD, 1932; MCILWAIN, 1959) and to participation of calcium in factors controlling membrane permeability (BRINK, 1954; SHANES, 1958). In this investigation, the calcium of mammalian cerebral cortex has been studied. The total calcium content of the tissue has been determined and the tissue permeability in media of varying calcium content estimated. Furthermore, the total tissue calcium has been measured under conditions which are known to alter the membrane permeability to sodium and potassium and the calcium exchanges has been determined under some of these conditions. M E T H O D S Media and tissue. Bicarbonate medium contained 120 mM-NaC1; 4.8 mM-KCl; 1.2 mM-KH,PO,; 1-3 mM-MgSO,; 10 niM-glucose; 26 mM-NaHCO, and varying CaCI, concentrations. The medium was equilibrated with O2 + CO, (95: 5, v/v) which was maintained in the vessels during incubation. * This investigation was supported by a Public Health Service Fellowship (Number BF-9,225-C3)

Studies in globoid (krabbe) leukodystrophy. ii. controlled thin-layer chromatographic studies of globoid body fractions in seven patients.

Abstract: THE EXACT pathochemical basis for globoid leukodystrophy has been somewhat unsettled in the literature (CUMINGS, 1960; AUSTIN, 1962, 1963). A significant part of this complex problem hinges on the question of what is contained in globoid bodies themselves. (Fig. 1). Although they are a hallmark of this disease, the few, scattered globoid bodies constitute only a very small percentage of devastated white matter. As a result, they have been assessed heretofore only by inference. The neurochemical aspects of this technical problem and various other aspects of globoid leukodystrophy are reviewed in detail elsewhere (AUSTIN, 19636, 1963~). The present report is therefore more limited in scope. Its chief purposes are twofold: (1) to describe a method by which a globoid body fraction may be separated from small amounts of white matter in enriched yields up to 22 per cent (2) to document the findings obtained in these globoid body fractions by thin-layer chromatography. Reference will also be made to a semiquantitative micromethod for estimating sphingolipids in these fractions. For purposes of brevity, the abbreviation GLDT is convenient and will be used here.

The distribution of acetylcholinesterase in peripheral nerves.

Abstract: IT IS generally assumed that under normal conditions, neuronal AChE* (acetylcholine acetyl-hydrolase, 3.1.1.7) is synthesized in the perikaryon. (see FELDBERG, 1957)t. Enzyme found in other parts of the neuron, in cell processes and on the presynaptic side of nerve endings must have been transported there from the cell bodies. The great length of axonal processes permits the study of AChE activity at various distances from cell bodies, thus providing some information concerning the character of the transport and the fate of the enzyme on its pathway. The peripheral nerves offer a more convenient material for such study than the tracts of central white matter. All cell bodies of axons contained in a nerve trunk are collected at one end of the nerve, not very far from one another. Thus, at each level of the nerve all axons are at similar distances from their origin and, in selected cases, from their endings as well. The Schwann cells probably do not contain AChE (CAVANAGH, THOMPSON and WEBSTER, 1954; TEWARI and BOURNE, 1960). There is some uncertainty on this point because of the residual AChE activity found in degenerating nerves after the disappearance of axons. In the dog it amounts to about 25 per cent of the original activity (unpublished). However, at that stage of degeneration, the number of Schwann cells has increased by about 8-fold (ABERCROMBIE and JOHNSON, 1946). In normal nerves the contribution of enzyme from Schwann cells to total activity presumably would be correspondingly weaker. Furthermore, as many behavioural and metabolic properties of Schwann cells are altered when their close association with the axon is disrupted (see LUBI~JSKA, 1961a, b) it is not impossible that the capability to synthesize A G E appears only after such disruption has taken place. Whether the Schwann cells in a normal nerve lack AChE activity or have so little that it cannot be detected by present histochemical methods, it seems safe to assume that their contribution to the total activity of the nerve trunk is insignificant and that AChE activity of the nerve represents almost exclusively the axonal activity. It was shown in preliminary work (LUBI~~SKA, NIEMIERKO, DERFELD and SZWARC, 1962) that in some peripheral nerves AChE activity decreases progressively in the proximo-distal direction.

The alkaline hydrolysis of the ethanolamine plasmalogen of brain tissue

Abstract: I N THE course of experiments on the enzymic breakdown of brain plasmslogens in uitro (ANSELL and SPANNER, 1963b) the method of DAWSON, HEMINGTON and DAVENPORT, (1962) was adopted for determining ethanolamine plasmalogen, which is the predominant plasmalogen in brain tissue. In this ingenious method, which stems from earlier work by the same group (DAWSON, 1954; DAWSON, 1960), the diacyl glycerophospholipids are completely deacylated by brief alkaline hydrolysis to yield watersoluble glycerylphosphoryl esters. The alkali-stable phospholipids are then heated with trichloroacetic acid (TCA)* solution containing mercuric chloride which liberates glycerylphosphoryl esters from the lysoplasmalogens. This method gives higher values for plasmalogens than the earlier (DAWSON, 1960) method since the formation of cyclic acetals during the acid hydrolysis of lysoplasmalogens is largely prevented. It was found that, even using this modification, the yield of glycerylphosphorylethanolamine (GPE) from the alkali-stable lipid on acid hydrolysis was much lower than would be expected from the amount of ethanolamine plasmalogen present in the tissue when this was independently determined from the vinyl ether and ethanolamine content of the lipids surviving the initial alkaline hydrolysis. These independent determinations gave values for total plasmalogen and ethanolamine plasmalogen which agreed more closely with those of RAPPORT and LERNER (1959) and WEBSTER (1960). In this paper the discrepancy is explained and the explanation exploited to prepare a native ethanolamine plasmalogen of approximately 90 per cent purity.

Studies of pyrimidine nucleotide metabolism in the central nervous system-i. metabolic effects and metabolism of 6-azauridine.

Abstract: INCORPORATION studies with [32P]phosphate (STRICKLAND, 1952 ; MANDEL et al., 1961), [14C]formate (MANELL and ROSSITER, 1955), [14C]adciiine, and [14C]orotic acid (KOENIG, 1958~7, 1959) have shown that RNA in nervous tissue is metabolically active. Purine and pyrimidine precursors are readily incorporated into the RNA of neurons, oligodendroglia, and other cell types (KOENIG, 19583, 1959, and unpublished). Observations that certain fluorinated pyrimidines, intrathecally administered, produce striking structural, physiological and biochemical alterations in brain and spinal cord of cat (KOENIG, 1958c, 1960,1962) have led us to pay particular attention to pyrimidine nucleotide metabolism in the neuraxis. In this communication we report results obtained with the riboside of 6-azauracil (as triazine-3,5-dione), a member of another class of pyrimidine analogues. This investigation was prompted, in part by reports that AzU, an agent which has antineoplastic effects in some animal tumors (JAFFEE et al., 1957; S ~ R M and KEILOVA, 1958), is markedly neurotoxic in man (WELLS et al., 1957; SHNIDER et al., 1960). Lethargy appears after several weeks of AzU ingestion, followed by hyperreflexia, mental obtundation, hallucinations and semicoma. Muscle weakness, involuntary movements, euphoria and other manifestations of neurological disturbance also may occur. Electroencephalographic abnormalities, consisting of a loss of fast rhythms, diminished response to photic stimulation, and generalized delta waves, precede the clinical disorder. This neuropsychiatric syndrome is slowly reversible when the drug is discontinued or the dose reduced. AzU and its riboside, AzUR, are believed to exert their carcinostatic effect by metabolic conversion to the monophosphate derivative, AzUMP, which in turn inhibits the enzyme orotidylic decarboxylase (HANDSCHUMACHER and PASTERNAK, 1958 ; PASTERNAK and HANDSCHUMACHER, 1959). This enzyme catalyzes the formation of UMP, the first naturally occurring pyrimidine nucleotide, from OMP, the ribonucleotide of OA. The OA pathway for the de nouo synthesis of pyrimidine nucleotides is